1. Field of the Invention
The present invention relates generally to gene expression in transgenic plants. More specifically, the present invention relates to unique enhancer elements from the tobacco rpL34 promoter which when operably linked to a promoter, increase the promoter""s rate of transcription and/or alter its activity in transformed plant cells.
2. Description of the Related Art
Given the technological advances in recombinant DNA technology made over the past decade it has become common practice to introduce new genetic material into plant cells, plant tissues or a whole plant to establish new traits that enhance the value of the plant or plant tissues. Both angiosperm and gymnosperm higher plants are included within the definition of xe2x80x9cplant.xe2x80x9d
A typical eukaroytic gene consists of a promoter region, introns, exons and a transcription terminator. The promoter region is typically located upstream of the transcribed region. The promoter determines the level and specificity of the gene transcription.
In eukaryotic organisms such as a plant, a promoter is not recognized directly by a RNA polymerase. Transcription initiation factors (TIFs) first bind to a promoter to form a preinitiation complex, and only then does an RNA polymerase bind to form an initiation complex.
A promoter for RNA polymerase II consists of a transcription initiation region, generally including a TATA box (the xe2x80x9cGoldberg-Hogness Boxxe2x80x9d) and frequently a CCAAT box, as well as upstream cis-acting elements. The transcription initiation region is also called the minimal promoter because it is the minimal DNA sequence required for a gene transcription. The TATA box directly binds a transcription factor complex that includes RNA polymerase II, for the initiation of DNA transcription. The TATA box is located approximately 25 base pairs upstream of the transcription start site. Further upstream, often between nucleotides xe2x88x9280 and xe2x88x92100, there can be a promoter element with homology to the consensus sequence CCAAT. Breathnach et al., Ann. Rev. Biochem. 50:349-383 (1981). In plants, the CCAAT box may be substituted by the AGGA box, at a similar distance from the transcription start site. Messing et al., in Genetic Engineering of Plants, Kosuge et al. Eds., pages. 211-227.
Promoters, together with enhancers and silencers, are cis-acting elements that control gene expression. Promoters are positioned next to the transcription start site and function in an orientation-dependent manner. Enhancer and silencer elements, which modulate the activity of promoters, may effect promoter activity in either orientation and at greater distances from the transcription start site. Khoury et al., Cell 33:3-13 (1983).
Enhancers can greatly increase the rate of transcription, and can generally function in either orientation and at various distances upstream or downstream from a given promoter. Enhancers may function in a wide variety of cells, or they may show strict cell or organism specificity. Enhancers may affect gene expression in response to environmental stimuli, such as illumination, nutrient concentration, heat shock, wounding, and anaerobiosis. These elements may also control gene expression in a development-specific, tissue-specific or tissue-preferred manner.
The prototype enhancer, the 72 bp tandem repeat of SV40 DNA, was initially identified as a cis-acting element located more than 100 base pairs upstream from the transcription initiation site of the early viral genes (Benoist et al., Nature 290:304-310 (1981); Gruss et al., Proc. Natl. Acad. Sci. USA 78:943-947 (1981). Deletion of this element reduced early gene expression by a factor of at least 100. Known cis-acting elements that enhance transcription in plants include a 16-bp palindrome sequence (5xe2x80x2-ACGTAAGCGCTTACGT-3) (SEQ ID NO: 4) derived from the octopine synthase gene (Ellis et al., EMBO J 6:3203 (1987) and the B-domain of the Cauliflower Mosaic Virus (CaMV) 35S promoter (Benfey et al. U.S. Pat. No. 5,097,025 (1992). A single and double base substitution or deletion in the ocs element caused significant loss in enhancer activity (Singh et al., Proc. Natl. Acad. Sci. USA 86:3733-3737 (1989). The mac gene promoter, which is composed of the mannopine synthase (mas) promoter from the Agrobacterium tumefaciens octopine Ti plasmid, and the B-domain of the 35S promoter, has much higher activity than that of the native 35S promoter, 35S promoter plus B-domain of the 35S promoter, or the mas promoter (Comai et al., Plant Mol. Biol. 15:373-381 (1990).
Synthesis of ribosomal proteins is universal in plant growth and development and represents an excellent model system for studying plant gene expression. Ribosomes are composed of a large (L) and a small (S) subunit built of RNA (r-RNAs) and proteins (rp) components.
Similar to other organisms, the tobacco genome contains various large subunit ribosomal protein (rpL) genes. One of these genes, referred to as rpL34, has been cloned and analyzed in some detail. Gao et al., Plant Mol Biol 25:761-770 (1994), Dai et al., Plant Mol. Biol. 32:1055-1065 (1996). The expression of the rpL34 gene is induced by plant wounding and chemicals and is meristem-specific.
Typically, higher levels of expression of mRNA is desired to increase the level of expression of the protein encoded by this mRNA. In addition, development-specific expression patterns enable protein production in plants during desired developmental stages, for example, post-harvest synthesis of foreign proteins. Also, tissue-specific patterns of expression enable novel schemes for utilization of non-crop plant portions for protein production as well as conferring necessary traits, such as disease resistance or chemical tolerance, to specific tissues. As recombinant DNA techniques are increasingly being applied to higher plants, there is an increased need for novel promoter elements to enable artificial regulation of gene expression. Specifically, there is a need for novel promoter elements that enable high levels of expression that is temporally, environmentally or developmentally controllable.
It is therefore an object of the present invention to provide novel promoter elements that enable high level gene expression. It is another object of the present invention to provide novel, non-specific enhancer elements which increase the activity of a promoter without affecting the promoter""s intrinsic specificity. The present invention also provides a method for increased gene expression at high levels in a temporally, environmentally or developmentally controlled manner.
In a first embodiment, the invention provides a promoter element consisting essentially of a polynucleotide molecule having the sequence of A which is xe2x88x92147 to xe2x88x92158 (SEQ ID NO:1) of FIG. 10, an enhancer element consisting essentially of a polynucleotide molecule having the sequence of B which is xe2x88x92159 to xe2x88x92181 (SEQ ID NO:2) of FIG. 10; an enhancer element consisting essentially of a polynucleotide molecule having the sequence of C which is xe2x88x92182 to xe2x88x92197 (SEQ ID NO:3) of FIG. 10.
In another embodiment, the invention provides a recombinant promoter construct having the general formula (I) or (II):
[(A)l, (B)m, (C)n, P]xe2x80x83xe2x80x83(I)
or
[(A)l, (B)m, (C)n]q, Pxe2x80x83xe2x80x83(II)
wherein A, B, and C are defined as above; and P is a native or non-native minimal promoter; and wherein A, B, C and P are operatively linked to each other and may be in any order; and wherein l, m, n are independent of each other and may be any integer between 0-5, and q is any integer between 1-5, provided that l, m and n are not simultaneously zero; and provided that the native configuration of the rpL34 promoter is excluded.
A preferred embodiment of the invention provides that P is selected from the group consisting of a constitutive promoter, a tissue-specific promoter and an inducible promoter.
Further embodiments of the invention provide a recombinant expression vector comprising the promoter elements, a plant cell comprising the expression vector, a transgenic plant regenerated from the cell and a method for producing a protein of interest using the transgenic plant.